Retroviruses are a group of viruses whose genetic material consists of single-stranded RNA. Following adsorption and entry of the retroviral RNA into the host cell, the viral RNA serves as a template for the synthesis of a complementary DNA strand. The DNA is then made double-stranded through the action of an enzyme having DNA polymerase activity; it is this double-stranded DNA which integrates into the host genome. The RNA-directed DNA polymerase activity responsible for the synthesis of complementary DNA from the viral RNA template is commonly called reverse transcriptase.
Retroviruses are of particular interest because a number of retroviruses have been implicated as the causative agents of various cancers, and other diseases. A retrovirus, human immunodeficiency virus, is the causal agent of acquired immunodeficiency syndrome (AIDS). Additionally, the reverse transcriptase enzymes themselves have become important reagents in molecular biology because of their ability to make complementary DNA from almost any RNA template. Thus, reverse transcriptase is commonly used to make nucleic acids for hybridization probes and to convert single-stranded RNA into a double-stranded DNA for subsequent cloning and expression.
Recently, reverse transcriptases have been used as a component of transcription-based amplification systems. These systems amplify RNA and DNA target sequences up to 1 trillion fold. See e.g., Burg et al., PCT Patent Application WO 89/01050 (1988); Gingeras et al., PCT Patent Application WO 88/10315 (1988); Davey and Malek, European Patent Application EPO 0329822 (1988); Gingeras et al., European Patent Application EPO 0373960 (1989); Malek and Davey, PCT Patent Application WO 91/02814 (1989); Kacian and Fultz, European Patent Application EPO 0408295 A2 (1990). All of these references are hereby incorporated by reference into this disclosure.
Some of the transcription-based amplification methods are exceptionally convenient since the amplification reaction according to these methods is isothermal. Thus, these systems are particularly suited for routine clinical laboratory use in diagnostic tests. Detection of pathogens causing infectious diseases and gene sequences associated with cancers or genetic diseases are among the most important uses of such tests. Reverse transcriptases are also employed as an initial step in some protocols when the polymerase chain reaction (PCR) is used to amplify an RNA target. See Malek et al., U.S. Pat. No. 5,130,238 (1992); and Mocharla et al., Gene 99:271-275 (1990). In such "RT-PCR" procedures, the reverse transcriptase is used to make an initial complementary DNA (cDNA) copy of the RNA target, which is then amplified by successive rounds of DNA replication.
The retroviral reverse transcriptases have three enzymatic activities: a RNA-directed DNA polymerase activity, a DNA-directed DNA polymerase activity, and an RNAse H activity. See Verma, The Reverse Transcriptase, Biochim. Biophys. Acta 473:1-38 (1977). The latter activity specifically degrades RNA contained in an RNA:DNA duplex. Degradation of the RNA strand of RNA:DNA intermediates by RNAse H is an important component of some transcription-based amplification systems and is to be distinguished from unwanted degradation due to contaminating nucleases, which interferes with amplification.
A disadvantage of the transcription-based amplification systems is their sensitivity to even trace amounts of nucleases. Since a number of important diseases may yield samples containing very few target nucleic acid molecules, the detection of small amounts of the target is often crucial for an accurate and timely diagnosis. Indeed, the value of target amplification methods is most important when the number of target molecules is low. At low input levels of the target nucleic acids, unwanted degradation of RNA targets or RNA or DNA reaction intermediates can lead to amplification failures and consequent misdiagnosis. Ribonuclease contamination is also a problem in RT-PCR reactions, since loss of the RNA target can lead to amplification failure.
Ribonucleases are relatively ubiquitous, and, in particular, are found in high concentrations in a variety of biological materials, including preparations of retroviruses and in cells commonly used to express recombinant proteins. Ribonucleases frequently contaminate reverse transcriptase preparations from a variety of sources and have been reported to interfere with synthesis of cDNAs, preparation of probes, and other uses besides target amplification alone. Often, an RNase inhibitor is included in the reaction to minimize the deleterious effects of this contamination. See e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual 8.11-8.13 (2d ed. Cold Spring Harbor Laboratory Press 1989), hereby incorporated by reference herein.
However, a number of substances commonly used to inhibit or inactivate RNAses, including detergents, chaotropes, organics, metals, proteases and metals are inappropriate for use in target amplification systems since they will inhibit the enzymes used for amplification as well. RNAse-inhibiting proteins such as human placental RNAse inhibitor, Blackburn et al., J. Biol. Chem. 252:5904 (1977) or rat liver RNAse inhibitor, Gribnau et al., Arch. Biochem. Biophys. 130:48-52 (1969), may be unstable, are expensive, and can contribute additional interfering substances such as nucleic acids and RNAses that are not inhibited by the inhibitor.
In addition to nucleases, traces of other enzymes, nucleic acids, and certain buffer salts may interfere with amplification reactions. While these substances are merely undesirable for many uses of reverse transcriptase, because of the nature of the amplification reaction it is critical that the enzyme preparation contain as low an amount of them as possible.
Isolation and purification of reverse transcriptase from various sources have been reported. In cases where the enzyme is isolated directly from virus particles, cells, or tissues, the cost is too high for widespread commercial use in diagnostic tests. See e.g., Kacian et al., Biochim. Biophys. Acta 46:365-83 (1971); Yang et al., Biochem. Biophys. Res. Comm. 47:505-11 (1972); Gerard, et al., J. Virol. 15:785-97 (1975); Liu et al., Arch. Virol. 55 187-200 (1977); Kato et al., J. Virol. Methods 9:325-39 (1984); Luke, et al. Biochemistry 29:1764-69 (1990); Le Grice et al., J. Virol. 65:7004-07 (1991). Additionally, these methods have not assured removal of substances that are significant inhibitors or contaminants that interfere with the use of reverse transcriptase for target amplification reactions. Another important consideration in the use of reverse transcriptases for a variety of purposes is the RNase H activity associated with the enzyme. The amount of RNase H activity and the way in which the RNase H activities work in coordination with the RNA- and DNA-dependent reverse transcriptase activities are important features affecting the utility of the enzyme for various purposes including transcription-based amplification systems. Too much or too little activity, the wrong kind of activity (such as non-specific RNases), or activities poorly coordinated with DNA synthesis can all lead to reduced performance in a particular application. Proper balance of the synthetic and degradative activities must be maintained; this is not only a function of the particular reverse transcriptase enzyme used, but also is dependent on the ability of the purification protocol to remove the RNA and/or DNA degrading activities.
The cloning and expression of reverse transcriptases in bacterial hosts has been previously reported. Attempts to clone and express reverse transcriptase from avian myeloblastosis virus (AMV-RT) did not lead to production of significant amounts of the purified enzyme. This is apparently due to the fact that the AMV-RT consists of two polypeptide chains, the .alpha. and .beta. chains, which must form a dimeric structure and undergo specific post-translational modifications in order to produce fully active enzyme. These same modifications do not occur when the gene is expressed in E. coli.
By contrast to the avian viral RTs, many reverse transcriptases derived from mammalian viruses consist of only one polypeptide chain; efforts to clone and express these enzymes have been more successful. In particular, Goff et al., U.S. Pat. No. 4,943,531 (1990) and Kotewicz et al., U.S. Pat. No. 5,017,492 have described methods for the purification of reverse transcriptase derived from Moloney Murine Leukemia Virus (MMLV-RT) and expressed in E. coli, which methods form the basis for the majority of commercial reverse transcriptase preparations.
Many commercial preparations of reverse transcriptase have been found unsuitable for use in target amplification and for other purposes due to nuclease contamination. See Sambrook, supra, previously incorporated by reference herein; Ryskov et al., Mol. Biol. Rep. 8:213-16 (1982). Other problems with commercial preparations of MMLV-RT may be related to an altered coordination between the DNA synthesis and RNAse H activities of the purified enzyme, reduced ability to bind and initiate synthesis at primer sites or to read through regions of tight secondary structure, or alternately may be due to DNase and other protein contamination. See Agronovsky, A. A., Anal. Biochem. 203:163-65 (1992). Additionally, commercial preparations made using the previously available methods for purification show significant lot-to-lot variability.
Moreover, due in part to the lengthy and labor-intensive purifications employed, the expense of the reagents and equipment employed for scale-up and the low yields of enzyme, the cost of such enzymes is prohibitive for their widespread commercial application in target amplification systems.
It is therefore an object of the present invention to provide an improved form of reverse transcriptase having the correct balance of DNA synthetic activities and RNAse H digestive activities, thereby being particularly suited for use in nucleic acid amplification methods.
It is another object of the present invention to provide a convenient source of reverse transcriptase containing low Levels of contaminants, such as undesired RNAses, that interfere with transcription-based amplification reactions by cloning and expressing a gene coding for an MMLV-RT enzyme having these properties in an E. coli host.
It is yet another object of the present invention to reduce the RNAse activity associated with the enzyme prior to and following purification by cloning and expressing the MMLV-RT gene in a ribonuclease-deficient strain of E. coli.
It is another object of the present invention to develop a simple purification scheme for the isolation of the enzyme.
It is a further object of the present invention to provide methods for the purification of the enzyme that achieve high levels of purity of RT at a low cost.